Rapid SDS-Gel capillary electrophoresis for the analysis of recombinant NADP(+)-dependent formate dehydrogenase during expression in Escherichia coli cells and its purification.
The level of expression in Escherichia coli cells and different steps of purification of the recombinant NADP(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions taken during the fermentation and purification process were analysed. The main advantages of SDS-Gel CE are short analysis time, high sensitivity, the possibility to quantify proteins at different ultraviolet wavelength, and small injection volumes. The data for each step of the fermentation process and during the purification were controlled by spectrophotometric analysis of enzyme activity and protein concentration as well as standard SDS PAGE. The molecular mass of the purified FDH was determined as 44,078 Da by matrix-assisted laser desorption/ ionisation time of flight mass spectrometry.
Klyushnichenko
V
V
Institute of Enzyme Technology, Heinrich-Heine University Düsseldorf. klyushni@ibt022.ibt.kfa-juelich.de
Tishkov
V
V
Kula
M R
MR
D002138:Calibration; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005560:Formate Dehydrogenases / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; C037212:formate dehydrogenase (NADP+); D005560:Formate Dehydrogenases